Despite its novelty in biological research, the involvement of RNA interference (RNAi) as a means of gene regulation has been established in numerous natural and biotechnology systems.(3,5-6,10,11,35) When perturbed in a cellular context, RNAi has been linked to diseases such as cancer;however, due to its general mode of action also shows promise as an adaptable therapeutic tool for a number of genetic diseases. While substantial progress has been made in this relatively young field, much remains unknown about the enzymatic mechanisms that underlie RNAi. This is especially evident in the case of Piwi RNA action, a more recently discovered class of RNAi which specifically regulates gene expression through the use of small RNAs ~24-31 nucleotides in length.(9-11) Bioinformatics analysis of the Piwi-related proteins has suggested key similarities and differences among individual members of this group including their mechanism of action. Structural information for Argonautes is rather limited, however, greatly hindering dissection of their modulated functions. Therefore, this proposal aims to determine the structural basis for RNA interference in the Piwi clade of Argonaute proteins. Given the strong structural component to the proposed research, the primary methods to be employed will be recombinant protein expression, purification, crystallization, and structure determination by X-ray diffraction. Once a structure solution has been determined, thorough biophysical and biochemical analysis of the protein will follow based on observations made from the structure. Overall, this research plan will elucidate many details of RNAi action and provide insight into RNA interference mechanisms. Given the links between RNAi, diseases such as cancer, and therapeutics, understanding the specifics of RNAi biology will enhance our understanding of the molecular basis of disease and assist in the development of more effective therapies. PUBLIC HEALTH RELEVANCE: This work will provide insight into the mechanism of RNA interference (RNAi) through biochemical and structural studies. Given the links between RNAi, diseases such as cancer, and therapeutics, understanding the specifics of RNAi biology will enhance our understanding of the molecular basis of disease and assist in the development of more effective therapies.